Gene silencing using mRNA-cDNA hybrids

ABSTRACT

The present invention provides novel compositions and methods for suppressing the expression of a targeted gene using mRNA-cDNA duplexes. The invention further provides novel methods and compositions for generating amplified mRNA-cDNA hybrids, whose quantity is high enough to be used for the invention&#39;s gene silencing transfection. This improved RNA-polymerase chain reaction method uses thermocycling steps of promoter-linked double-stranded cDNA or RNA synthesis, in vitro transcription and then reverse transcription to amplify the amount of mRNA-cDNA hybrids up to two thousand folds within one round of the above procedure.

RELATED APPLICATION DATA

This applications claims priority to U.S. provisional application Ser.No. 60/222,479, filed Aug. 2, 2000, the entire disclosure of which isincorporated herein by this reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.CA083716 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to gene silencing phenomenon,and particularly to gene silencing using mRNA-cDNA hybrids and methodsfor generating mRNA-cDNA hybrids for use in gene silencing.

BACKGROUND OF THE INVENTION

Gene silencing or inhibiting the expression of a gene holds greattherapeutic and diagnostic promise. An example of this approach isantisense technology which can be used to inhibit gene expression invivo. However, many problems remain with development of effectiveantisense technology. For example, DNA antisense oligonucleotidesexhibit only short term effectiveness and are usually toxic at the dosesrequired. Similarly, the use of antisense RNAs has also provedineffective due to stability problems.

Other approaches to quelling specific gene activities areposttranscriptional gene silencing (PTGS) and RNA interference (RNAi)phenomena, which have been found capable of suppressing gene activitiesin a variety of in-vivo systems, including plants (Grant, S. R. (1999)Cell 96, 303-306), Drosophila melanogaster (Kennerdell, J. R. andCarthew, R. M. (1998) Cell 95, 1017-1026, Misquitta, L. and Paterson, B.M. (1999) Proc. Natl. Acad. Sci. USA 96, 1451-1456, and Pal-Bhadra, M.,Bhadra, U., and Birchler, J. A. (1999) Cell 99, 35-46), Caenorhabditiselegans (Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok,A., and Timmons, L. (1999) Cell 99, 123-132, Ketting, R. F., Haverkamp,T. H., van Luenen, H. G., and Plasterk, R. H. (1999) Cell 99, 133-141,Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., andMello, C. C. (1998) Nature 391, 806-811 and Grishok, A., Tabara, H., andMello, C. C. (2000) Science 287, 2494-2497), zebrafish (Wargelius, A.,Ellingsen, S., and Fjose, A. (1999) Biochem. Biophys. Res. Commun. 263,156-161) and mouse (Wianny, F. and Zernicka-Goetz, M. (2000) Nature CellBiol. 2, 70-75). In general, the transfection of a plasmid-like DNAstructure (transgene) into cells induces PTGS phenomena, while that of adouble-stranded RNA (ds-RNA) causes an RNAi effect.

These phenomena appear to evoke an intracellular sequence-specific RNAdegradation process, affecting all highly homologous transcripts, calledcosuppression. It has been proposed that such cosuppression results fromthe generation of small RNA products (21˜25 nucleotide bases) by anRNA-directed RNA polymerase (RdRp) (Grant supra) and/or a ribonuclease(RNase) (Ketting et al. supra, Bosher, J. M. and Labouesse, M. (2000)Nature Cell Biology 2, 31-36 and Zamore, P. D., Tuschl, T., Sharp, P.A., and Bartel, D. P. (2000) Cell 101, 25-33.) activity on an aberrantRNA template, derived from the transfecting nucleic acids or viralinfection. Although an RdRp-independent endoribonucleolysis model hasbeen proposed for the RNAi effect in Drosophila (Zamore, et al. supra),the RdRp homologues were widely found in Arabidopsi thalianas asSde-1/Sgs-2 (Yang, D., Lu, H., and Erickson, J. W. (2000) CurrentBiology 10, 1191-1200), Neurospora crassa as Qde-1 (Cogoni, C. andMacino, G. (1999) Nature 399, 166-169) and Caenorhabditis elegans asEgo-1 (Smardon, A., Spoerke, J. M., Stacey, S. C., Klein, M. E., Mackin,N., and Maine, E. M. (2000) Curr. Biol. 10, 169-171). Thus, RdRphomologues appear to be a prerequisite for maintaining along-term/inheritable PTGS/RNAi effect (Bosher, et al. supra).

Although PTGS/RNAi phenomena appear to offer a potential avenue forinhibiting gene expression, they have not been demonstrated to work wellin higher vertebrates and, therefore, their widespread use in highervertebrates is still questionable. Consequently, there remains a needfor an effective and sustained method and composition for inhibitinggene function in vivo in higher vertebrates.

SUMMARY OF THE INVENTION

The present invention provides a novel composition and method forinhibiting gene function in prokaryotes and eukaryotes in vivo and invitro. Without being bound by any particular theory, this methodpotentially is based on an RdRp-dependent gene silencing phenomenon,similar to PTGS/RNAi, which is hereafter termed DNA-RNA interference(D-RNAi). In accordance with the present invention, mRNA-cDNA hybridsare used for inhibiting gene function. The mRNA-cDNA hybrids of thepresent invention can be used to target a gene selected from the groupconsisting of pathogenic nucleic acids, viral genes, mutated genes, andoncogenes.

In specific embodiments, the present invention provides a compositionfor inhibiting the expression of a targeted gene in a substrate whereinthe composition comprises an mRNA-cDNA hybrid. The composition of theinvention is effective to inhibit the expression of the targeted gene invitro or in vivo. The mRNA-cDNA hybrid may be synthesized using themethod described below. In one embodiment, the mRNA of the invention'shybrids is comprised of part or all of the unspliced mRNA transcript ofthe targeted gene. In another embodiment, the mRNA is comprised of partor all of the spliced mRNA transcript of the targeted gene. In yetanother embodiment, the mRNA is comprised of a combination of part orall of the spliced and unspliced mRNA transcript of the targeted gene.To prepare the composition, the mRNA-cDNA hybrid may be prepared bycomplementarily combining the sense-oriented mRNA of one of the threepreceding embodiments with its corresponding antisense-oriented cDNAmolecule in a base-pairing double stranded form.

The composition of the invention may be used to treat a substrate thatis a cell or an organism, which may be either prokaryotic or eukaryotic.

The composition of the invention may be administered to the substrateusing methods and compositions known to one of ordinary skill in theart. For example, the composition of the invention may further comprisea carrier molecule, which is capable of being taken up by a cell. Thecompositions may be administered orally, intravenously, transdermally,etc.

Further, the present invention provides a method for inhibiting theexpression of a targeted gene in a substrate, comprising the steps of:a) providing a composition comprising an mRNA-cDNA hybrid capable ofinhibiting the expression of the targeted gene in the substrate and b)contacting the substrate with the composition under conditions such thatgene expression in the substrate is inhibited. The substrate can expressthe targeted gene in vitro or in vivo. The composition to be used inthis method of the invention may be a composition selected from one ofthe above-described compositions.

In one embodiment, the mRNA-cDNA hybrid targets a gene selected from thegroup consisting of pathogenic nucleic acids, viral genes, mutatedgenes, and oncogenes. In another embodiment, the mRNA-cDNA hybridinhibits β-catenin expression. In yet another embodiment, the mRNA-cDNAhybrid inhibits bcl-2 expression. In various embodiments, the substrateis a prokaryote, e.g., a virus or a bacterial cell, or a eukaryote orthe cell of a eukaryote. Eukaryotes contemplated by the inventioninclude, without limitation, vertebrates, e.g., mice, chimpanzees andhumans.

The invention also provides compositions and methods for prepairingmRNA-cDNA hybrids. Specifically, the present invention provides methodsfor generating mRNA-cDNA hybrids, comprising the steps of: a) providing:i) a solution comprising a nucleic acid template, ii) one or moreprimers sufficiently complementary to the sense conformation of thenucleic acid template, and iii) one or more promoter-linked primerssufficiently complementary to the antisense conformation of the nucleicacid template, and having an RNA promoter; b) treating the nucleic acidtemplate with one or more primers under conditions such that a firstcDNA strand is synthesized; c) treating the first cDNA strand with oneor more promoter-linked primers under conditions such that apromoter-linked double-stranded nucleic acid is synthesized; d) treatingthe promoter-linked double-stranded nucleic acid under conditions suchthat essentially mRNA fragments are synthesized; and e) treating mRNAfragments with one or more primers under conditions such that anmRNA-cDNA hybrid is synthesized. The methods of the present inventioncan comprise the step of repeating steps b) through e) for a sufficientnumber of cycles to obtain a desired amount of amplified product.

The treating step in step b) can comprise heating the solution at atemperature above 90° C. to provide denatured nucleic acids. Thetreating step in step c) can comprise treating the first cDNA strandwith one or more promoter-linked primers at a temperature ranging fromabout 35° C. to about 75° C. The treating step in step c) can alsocomprise treating the EDNA strand with one or more promoter-linkedprimers in the presence of a polymerase.

In one embodiment, the polymerase is selected from the group consistingof DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, RNApolymerases, Taq-like DNA polymerase, TTh-like DNA polymerase, C. therm.polymerase, viral replicases, and combinations thereof. The viralreplicases can be selected from the group consisting of avianmyeloblastosis reverse transcriptase and Moloney murine leukemia virusreverse transcriptase. In particular, the AMV reverse transcriptase doesnot have RNase activity.

The treating step in step d) can comprise treating the promoter-linkeddouble-stranded nucleic acid with an enzyme having transcriptaseactivity at about 37° C. The enzyme having transcriptase activity can beselected from the group consisting of RNA polymerases and viralreplicases. The RNA polymerases can be selected from the groupconsisting of T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase,and M13 RNA polymerase.

The primers are complementary to the 3′-ends of the sense conformationof the nucleic acid template. In one embodiment, one or more primerscomprise a poly(dT)₂₄ primer.

The promoter-linked primers are complementary to the 5′-ends of theantisense conformation of the nucleic acid template. In one embodiment,one or more promoter-linked primers comprise oligo(dC)₁₀N-promoterprimers. The oligo(dC)₁₀N-promoter primers can also compriseoligo(dC)₁₀G-T7 primers, oligo(dC)₁₀A-T7 primers, oligo(dC)₁₀T-T7primers, and combinations thereof. The promoter-linked double-strandednucleic acid can be selected from the group consisting ofpromoter-linked double-stranded DNAs and promoter-linked double-strandedRNAs.

In one embodiment, the treating step in step e) comprises treating mRNAfragments with one or more primers at a temperature ranging from about35° C. to about 75° C.

The methods of the present invention can further comprise the step ofincorporating one or more nucleotide analogs into the cDNA portion ofthe mRNA-cDNA hybrid to prevent degradation. In another embodiment, themethods of the present invention further comprise the step of contactingmRNA-cDNA hybrids with a reagent for gene knockout transfection. Thereagent can be selected from the group consisting of chemicaltransfection reagents and liposomal transfection reagents.

The present invention relating to mRNA-cDNA gene knockout technology canbe used as a powerful new strategy in the field of antisense genetherapy. The strength of this novel strategy is in its low dose,stability, and potential long-term effects. Applications of the presentinvention include, without limitation, the suppression of cancer relatedgenes, the prevention and treatment of microbe related genes, the studyof candidate molecular pathways with systematic knock out of involvedmolecules, and the high throughput screening of gene functions based onmicroarray analysis, etc. The present invention can also be used as atool for studying gene function in physiological conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of the RNA-PCR method for RNAamplification.

FIG. 2 shows a schematic representation of experimental procedures fortesting interference of bcl-2 gene expression in androgen-treated humanprostate cancer LNCaP cells, according to one embodiment of the presentinvention.

FIG. 3 shows different templates for bcl-2 gene interference, accordingto one embodiment of the present invention.

FIG. 4 shows a proposed model for long-term PTGS/RNAi/D-RNAi mechanisms.

FIG. 5 shows a linear plot of the interaction between incubation timeand cell growth number in the methods of the present invention.

FIG. 6 shows potential D-RNAi-related RdRp enzymes by differentα-amanitin sensitivity.

FIG. 7 shows a schematic representation for producing mRNA-cDNA hybrids.

FIG. 8 shows Northern results of blank control and mRNA-cDNA hybrid inone embodiment of the present invention.

FIG. 9 shows the effect of in vivo delivery of mRNA-cDNA hybrid ontargeted gene expression, in one embodiment of the present invention.FIG. 9A shows the embryo prior to microinjection; FIG. 9B shows theembryo after injection. FIG. 9C shows the Northern analyses resultsafter treatment with mRNA-cDNA hybrid, while FIG. 9D shows the liposomecontrol embryos.

FIG. 10 illustrates the suppression of β-catenin, according to oneembodiment of the present invention.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acid bases are matched according tothe base pairing rules. Alternatively, complementarity may be “complete”or “total” between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as indetection methods which depend upon binding between nucleic acids.

As used herein, the term “template” refers to a nucleic acid moleculebeing copied by a nucleic acid polymerase. A template can besingle-stranded, double-stranded or partially double-stranded, dependingon the polymerase. The synthesized copy is complementary to thetemplate, or to at least one strand of a double-stranded or partiallydouble-stranded template. Both RNA and DNA are synthesized in the 5′ to3′ direction. The two strands of a nucleic acid duplex are alwaysaligned so that the 5′ ends of the two strands are at opposite ends ofthe duplex (and, by necessity, so then are the 3′ ends).

As used herein, the term “mRNA” or “messenger RNA” refers to a singlestranded RNA molecule that is synthesized during transcription, iscomplementary to one of the strands of double-stranded DNA, and servesto transmit the genetic information contained in DNA to the ribosomesfor protein synthesis. The mRNA may be spliced, partially spliced orunspliced, and may be eukaryotic or prokaryotic mRNA.

As used herein, the term “nucleic acid template” refers to adouble-stranded DNA/RNA, a single-stranded DNA, an mRNA/aRNA or anRNA-DNA hybrid.

As used herein, the term “poly (dT)n promoter sequence” refers to an RNApolymerase promoter sequence coupled with a poly-deoxythymidylate (dT)sequence at its 3′ end, in which the number n of linked dTs lies in therange of about five to about thirty and most preferably is about twentysix.

As used herein, the term “oligo(dC)nN-T7 primer” refers to an RNA primercoupled with a poly-deoxycytidylate (dC). The number n of totalincorporated nucleotides is in the range of about 5 to about 30 and ismost preferably about 12.

As used herein, the term “primer” refers to an oligonucleotidecomplementary to a template. The primer complexes with the template togive a primer/template complex for initiation of synthesis by a DNApolymerase. The primer/template complex is extended by the addition ofcovalently bonded bases linked at its 3′ end, which are complementary tothe template in DNA synthesis. The result is a primer extension product.Virtually all known DNA polymerases (including reverse transcriptases)require complexing of an oligonucleotide to a single-stranded template(“priming”) to initiate DNA synthesis.

As used herein, the term “promoter-linked primer” refers to anRNA-polymerase-promoter sense sequence coupled with a gene-specificcomplementary sequence in its 3′-end for annealing to the antisenseconformation of a nucleic acid template.

As used herein, the term “DNA-dependent DNA polymerase” refers to anenzyme that synthesizes a complementary DNA copy from a DNA template.Examples are DNA polymerase I from E. coli and bacteriophage T7 DNApolymerase. Under suitable conditions a DNA-dependent DNA polymerase maysynthesize a complementary DNA copy from an RNA template.

As used herein, the terms “DNA-dependent RNA polymerase” and“transcriptase” refer to enzymes that synthesize multiple RNA copiesfrom a double-stranded or partially-double stranded DNA molecule havinga promoter sequence. Examples of transcriptases include, but are notlimited to, DNA-dependent RNA polymerase from E. coli and bacteriophagesT7, T3, and SP6.

As used herein, the terms “RNA-dependent DNA polymerase” and “reversetranscriptase ” refer to enzymes that synthesize a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template. Thus,reverse transcriptases are both RNA-dependent and DNA-dependent DNApolymerases. As used herein, the term “RNAse H” refers to an enzyme thatdegrades the RNA portion of an RNA/DNA duplex. RNAse H's may beendonucleases or exonucleases. Most reverse transcriptase enzymesnormally contain an RNAse H activity in addition to their polymeraseactivity. However, other sources of the RNAse H are available without anassociated polymerase activity. The degradation may result in separationof RNA from a RNA/DNA complex. Alternatively, the RNAse H may simply cutthe RNA at various locations such that portions of the RNA melt off orpermit enzymes to unwind portions of the RNA.

As used herein, the terms “hybridize” and “hybridization” refer to theformation of complexes between nucleotide sequences which aresufficiently complementary to form complexes via Watson-Crick basepairing. Where a primer (or splice template) “hybridizes” with target(template), such complexes (or hybrids) are sufficiently stable to servethe priming function required by the DNA polymerase to initiate DNAsynthesis.

As used herein, the term “sense conformation” refers to a nucleic acidsequence in the same sequence order and composition as its homolog mRNA.

As used herein, the term “antisense conformation” refers to a nucleicacid sequence complementary to its respective mRNA homologue. Theantisense RNA (aRNA) refers to a ribonucleotide sequence complementaryto an mRNA sequence in an A-U and C-G composition, and also in thereverse orientation of the mRNA.

As used herein, the term “gene” refers to a nucleic acid (e.g., DNA)sequence that comprises coding sequences necessary for the production ofa polypeptide or precursor. The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional properties (e.g., enzymaticactivity, ligand binding, signal transduction, etc.) of the full-lengthor fragment are retained. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “interveningregions” or “intervening sequences.”

As used herein, the term “gene silencing” refers to a phenomenon wherebya function of a gene is completely or partially inhibited. Throughoutthe specification, the terms “silencing,” “inhibition,” “quelling,”“knockout” and “suppression,” when used with reference to geneexpression or function, are used interchangeably.

As used herein, the term “oligonucleotide” is defined as a moleculecomprised of two or more deoxyribonucleotides or ribonucleotides,preferably more than three, and usually more than ten. The exact sizewill depend on many factors, which in turn depends on the ultimatefunction or use of the oligonucleotide. The oligonucleotide may begenerated in any manner, including chemical synthesis, DNA replication,reverse transcription, or a combination thereof.

As used herein, the term “transfection” refers to the introduction offoreign DNA into eukaryotic cells. Transfection can be accomplished by avariety of means known to the art, including, but not limited to,calcium phosphate-DNA co-precipitation, DEAE-dextran-mediatedtransfection, polybrene-mediated transfection, electroporation,microinjection, liposome fusion, lipofection, protoplast fusion,retroviral infection, and biolistics.

A primer is selected to be “substantially” or “sufficiently”complementary to a strand of specific sequence of the template. A primermust be sufficiently complementary to hybridize with a template strandfor primer elongation to occur. A primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being substantially complementaryto the strand. Non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the template tohybridize and thereby form a template primer complex for synthesis ofthe extension product of the primer.

As used herein, the term “amplification” refers to nucleic acidreplication involving template specificity. Template specificity isfrequently described in terms of “target” specificity. Target sequencesare “targets” in the sense that they are sought to be sorted out fromother nucleic acid. Amplification techniques have been designedprimarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that will processonly specific sequences of nucleic acid in a heterogeneous mixture ofnucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is thespecific template for the replicase (Kacian et al., Proc. Natl. Acad.Sci. USA 69:3038 (1972)). Other nucleic acid will not be replicated bythis amplification enzyme. Similarly, in the case of T7 RNA polymerase,this amplification enzyme has a stringent specificity for its ownpromoters (Chamberlin et al., Nature 228:227 (1970)). Taq and Pfupolymerases, by virtue of their ability to function at high temperaturedisplay high specificity for the sequences bounded, and thus defined bythe primers.

As used herein, the terms “amplifiable nucleic acid” and “amplifiedproducts” refer to nucleic acids which may be amplified by anyamplification method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides) which is capable of hybridizing to anotheroligonucleotide of interest, whether occurring naturally as in apurified restriction digest or produced synthetically, recombinantly orby PCR amplification. A probe may be single-stranded or double-stranded.Probes are useful in the detection, identification and isolation ofparticular gene sequences.

As used herein, the term “polymerase chain reaction” (“PCR”) refers to amethod for increasing the concentration of a segment in a targetsequence from a mixture of genomic DNA without cloning or purification(U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, hereby incorporatedby reference). This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target it sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i e., denaturation, annealing andextension constitute one “cycle ”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction ” (“PCR”). Because thedesired amplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., incorporation of biotinylated primers followed byavidin-enzyme conjugate detection; incorporation of ³²P-labeleddeoxynucleotide triphosphates, such as dCTP or dATP, into the amplifiedsegment). In addition to genomic DNA, any oligonucleotide orpolynucleotide sequence can be amplified with the appropriate set ofprimer molecules. In particular, the amplified segments created by thePCR process itself are, themselves, efficient templates for subsequentPCR amplifications.

As used herein, the term “portion” when in reference to a protein ornucleic acid sequence refers to fragments of that protein or nucleicacid sequence. Fragments of a protein can range in size from four aminoacid residues to the entire amino acid sequence minus one amino acid.

The term “nucleotide analog” as used herein refers to modified ornon-naturally occurring nucleotides such as 7-deaza purines (i.e.,7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogsand comprise modified forms of deoxyribonucleotides as well asribonucleotides.

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size, followed by transfer of the RNA from the gel to asolid support such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (Sambrook et al., Molecular Cloning, 2^(nd)Ed., Cold Spring Harbor Laboratory Press, pp 7.39-7.52 (1989)).

As used herein, the term “Southern blot” refers to the analysis of DNAon agarose or acrylamide gels to fractionate the DNA according to size,followed by transfer of the DNA from the gel to a solid support such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists(Sambrook et al., supra).

The term “virus” refers to obligate, ultramicroscopic, intracellularparasites incapable of autonomous replication (i.e., replicationrequires the use of the host cell's machinery).

As used herein, the terms “Taq-like polymerase” and “Taq polymerase”refer to Taq DNA polymerase and derivatives. Taq DNA is widely used inmolecular biology techniques including recombinant DNA methods. Forexample, various forms of Taq have been used in a combination methodwhich utilizes PCR and reverse transcription (See e.g., U.S. Pat. No.5,322,770, incorporated herein in its entirety by reference). DNAsequencing methods which utilize Taq DNA polymerase have also beendescribed. (See e.g., U.S. Pat. No. 5,075,216, incorporated herein inits entirety by reference).

As used herein, the terms “TTh-like polymerase” and “TTh polymerase”refer to polymerase isolated from Thermus thermophilus. Tth polymeraseis a thermostable polymerase that can function as both reversetranscriptase and DNA polymerase (Myers and Gelfand, Biochemistry30:7662-7666 (1991)). It is not intended that the methods of the presentinvention be limited to the use of Taq-like or TTh-like polymerases.Other thermostable DNA polymerases which have 5′ to 3′ exonucleaseactivity (e.g., Tma, Tsps17, TZ05, Tth and Taf) can also be used topractice the compositions and methods of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for generatingmRNA-cDNA hybrids and compositions and methods using the same for genesilencing. It is proposed that the mRNA-cDNA hybrids effect anRdRp-dependent gene silencing phenomenon, i.e., DNA-RNA interference(D-RNAi). The advantages of using D-RNAi instead of ds-RNA are asfollows: 1) the cDNA part of a D-RNAi can be modified bynucleotide-analog incorporation to increase the stability andeffectiveness of transfected probe activities; 2) the RdRp enzyme mayprovide higher affinity to the mRNA template of a D-RNAi compared to ads-RNA due to lower binding interaction between DNA-RNA duplexes thanRNA-RNA duplexes; and 3) the cDNA part of a D-RNAi provides furtherantisense gene knockout activity in addition to the PTGS gene silencingmechanisms of the sense-RNA template, resulting in multiple specificgene interference effects with one probe.

The m-RNA-cDNA hybrids of the invention are preferably prepared using animprovement of the so-called RNA-PCR described in U.S. Pat. No.6,197,554.

RNA-PCR

A PCR-like reaction performed on mRNAs, named the RNA-polymerase chainreaction (RNA-PCR), to provide a highly efficient amplification(˜250-fold/cycle) of the whole mRNA repertoire exists in the prior art.(Lin et al., Nucl. Acids. Res. 27:4585-4589 (1999)); U.S. Pat. No.6,197,554 to Lin et al., incorporated herein by reference in theirentirety). The elevated thermocycling temperature prevents rapiddegradation of short-lived mRNAs and further reduces the secondarystructure of mRNAs to increase the accessibility of enzyme interactionsand the production of more complete full-length mRNAs. The procedureuses thermostable enzymes, including Tth-like DNA polymerases withreverse transcriptase activity and thermostable RNA polymerases. The useof proofreading RNA polymerases for amplification not only provideshigher fidelity but also eliminates preferential amplification ofabundant mRNA species. Additionally, rapid and simple cell fixation andpermeabilization steps inhibit any alterations in gene expression duringspecimen handling or genomic contamination. (See, Embleton et al., Nucl.Acids Res. 20: 3831-3837 (1992)).

The procedure, depicted in FIG. 1, is as follows: (1) prevention of mRNAdegradation; (2) first reverse transcription; (3) a tailing reaction toadd 5′-poly(dT) and 3′-poly(dG) to the first strand cDNAs; (4)denaturation and then cDNA double-stranding by the extension of anoligo(dC)-promoter primer complementary to the 3′-poly(dG) tail; (5)promoter-driven transcription to amplify mRNAs up to 2000-fold in onecycle; (6) repeating steps 2, 4 and 5 (without 3) to achieve the desiredmRNA amplification.

The procedure can be implemented using a poly(dT)₂₄ primer to generatethe first-strand cDNAs. Another oligo(dC)-promoter primer is used togenerate the second-strand cDNAs. Both strands together form thepromoter-linked double-stranded cDNAs from the original mRNAs. Theoligo(dC)-promoter primer is an equal mixture of oligo(dC)₁₀N sequences(N=dG, dA or dT) coupled to an RNA promoter for in vitro transcriptionalong the doublestranded cDNA templates. Because the promoter region isincorporated in the 5′-end of the second-strand cDNAs which has the samesequence and composition as the original mRNAs, the transcriptionproducts are all in the form of mRNAs, not aRNAs. These amplified mRNAsnot only share the same properties but also have the full integrity oftheir original mRNAs, depending on the quality of the firstpromoter-linked double-stranded cDNAs.

Methods for Generating mRNA-cDNA Hybrids for Gene Silencing

The present invention provides a simple, fast, and inexpensive methodfor amplifying specific mRNA-cDNA hybrids for gene silencingtransfection. The mRNA-cDNA hybrids can be used for screening specialgene functions, for manipulating gene expressions in vitro, and fordesigning a therapy for genetic diseases in vivo.

The present invention is directed to an improved RNA-polymerase chainreaction method for generating mRNA-cDNA hybrid duplexes (FIG. 7) forgene interference effects in cells (“D-RNAi”).

An improved RNA-PCR method that uses a gene-specific primer andpromoter-primer in a thermocycling procedure to amplify specificmRNA-cDNA sequences is provided. This thermocycling procedure preferablystarts from reverse transcription of mRNAs with Tth-like polymerases,following a promoter-incorporation and cDNA double-stranding reactionwith the same Tth-like polymerases. The resulting promoter-linkeddouble-stranded DNAs serve as transcriptional templates for amplifyingmRNA up to 2000 fold/cycle by RNA polymerases. Alternatively, RNAreplicases can be used to directly amplify mRNA when the startingtemplates are aRNAs or ds-RNAs containing a functional recognition sitefor the replicases. The thermocycling procedure can be repeated for moreamplification to a desired amount of mRNA-cDNA hybrids.

The amplification cycling procedure of the present invention presentsseveral advantages over prior amplification methods. First, D-RNAiprobes from low-copy rare mRNA species can be prepared within threerounds of amplification cycling without misreading mistakes. Second, themRNA-cDNA hybrid amplification is linear and does not result inpreferential amplification of nonspecific gene sequences. Third, mRNAdegradation is inhibited by thermostable enzymatic conditions withoutRNase activities. Finally, the use of RNase H activity is restricted,thereby preserving the integrity of final mRNA-cDNA constructs. Unlikecurrent NASBA methods (Compton, Nature 350: 91-92 (1991)), this improvedRNA-PCR procedure contains no RNase H activity which can degrade the RNAstructure of a RNA-DNA hybrid. Thus, the methods of the presentinvention can be used to prepare high amounts of pure and specificmRNA-cDNA hybrids for transducing biological effects of interest invitro as well as in vivo.

The labeling of mRNA-cDNA hybrids can be accomplished by incorporationof labeled nucleotides or analogs during reverse transcription ofTth-like polymerase activity. The mRNA-cDNA hybrids of the presentinvention can be used as probes in a variety of applications, includingbut not limited to, Northern blots, Southern blots, dot hybridization,in situ hybridization, position cloning, nucleotide sequence detection,and antisense knockout transfection. The mRNA-cDNA structures can alsocomprise nucleotide analogs to prevent degradation, resulting in morestability and effectiveness of the probe transfection. In addition tothe gene silencing effects (or PTGS) caused by the mRNA part, theantisense cDNA part of the mRNA-cDNA hybrids can further providetraditional gene knockout schemes through the binding of itself tointracellular mRNA homologues. Both gene knockout effects ensure thesuccess of degrading certain specific mRNA species in cells, causing abroad and multiple gene interference result better than previousantisense gene knockout methodology.

Although certain preferred embodiments of the present invention havebeen described, the spirit and scope of the invention is by no meansrestricted to what is described above. For example, different nucleicacid templates, as well as different specific primers for reversetranscription and polymerase extension reaction can be used to practicethe methods of the present invention. Furthermore, differentpromoter-linked primers for transcription can also be used to practicethe methods of the present invention. Moreover, different thermostableenzymes can be used to practice the methods of the present invention.

Gene Silencing Using mRNA-cDNA Hybrids: In Vitro Prostrate Cancer Model

As noted earlier, posttranscriptional gene silencing (PTGS) and RNAinterference (RNAi) have been found capable of quelling specific geneactivities in a variety of in vivo systems.

According to the invention provided herein, ectopic transfection of asequence-specific messenger RNA (mRNA)-complementary DNA (cDNA) hybrid(instead of a transgene or ds-RNA) is used to induce intracellular genesilencing in human cells. Although previous transgene/ds-RNAtransfection experiments showed that PTGS/RNAi effects are limited toplants and some simple animals, using the present invention, specificgene interference in higher eukaryotes, e.g. of bcl-2 expression inhuman LNCaP prostate cancer cells, using the D-RNAi has beensuccessfully demonstrated.

Normal human prostatic secretory epithelial cells do not express bcl-2protein, whereas neoplastic prostate tissues from androgen-ablationpatients show an elevated level of this apoptosis-suppressingoncoprotein. It is known in the art that over-expression of bcl-2protects prostate cancer cells from apoptosis in vitro, and confersresistance to androgen depletion in vivo. The tumorigenic and metastaticpotentials of LNCaP cells are also significantly increased after bcl-2stimulation by either androgen or transgene treatment. Such inhibitionof apoptosis can be blocked by treatment with bcl-2 antisenseoligonucleotides, but many apoptotic stimuli such as etoposide orphorbol ester cannot be blocked.

The potential utility of D-RNAi in preventing bcl-2 expression wastherefore tested on androgen-stimulated LNCaP cells, expecting toincrease cancer cell susceptibility to apoptotic stimuli and reducetumorigenic outgrowth in vitro. Following previous findings, LNCaP cellswere treated with dihydrotestersterone (100 nM 5α-anrostan-17β-ol-3-one)to block the apoptotic effect of phorbol ester (10 nMphorbol-12-myristate-13-acetate). When treated with the methods andcompositions of this invention LNCaP cells induced an anti-bcl-2 D-RNAieffect to resume the apoptosis of the androgen- and phorbolester-treated cancer cells (FIG. 2).

The identification of D-RNAi in human prostate cancer LNCaP cells is abreakthrough in the development of evolutionary gene silencing effectsin higher level animal systems. When using RNA-DNA hybrids larger than500 bases, a significant long-term (>6 days) PTGS/RNAi-like genesilencing effects in the mRNA-cDNA transfected cells was detected afteran average 36-hour incubation with only one transfection.

FIG. 3 shows the analysis of different templates for bcl-2 geneinterference, namely: (1) blank control; (2) mRNA-cDNA hybrid; (3)aRNA-cDNA hybrid; and (4) ds-RNA in LNCaP cells. FIG. 3A shows changesof cell proliferation rate and morphology. Chromosomal DNAs were stainedby propidium iodide. Although the ds-RNA transfection also showed minormorphological changes, a significant cell growth inhibition andchromosomal condensation only occurred in the mRNA-cDNA transfection(n=4). FIG. 3B shows genomic laddering patterns demonstrating apoptosisinduction by the bcl-2 mRNA-cDNA transfection. FIG. 3C presents Northernblots showing a strong gene silencing effect of the mRNA-cDNAtransfection in bcl-2 expression. As shown in FIG. 3, the transfectionof bcl-2 mRNA-cDNA hybrids (5 nM) into LNCaP cells was sufficient tosilence bcl-2 expression and cause apoptosis (chromosomal condensationand genomic DNA laddering fragmentation), which have not been foundusing double-stranded DNA (ds-RNA) or aRNA-cDNA hybrid transfections.

Each transfection of the antisense DNA probes provides a fast (within24-hour incubation) and relatively short-term (2 to 3 days) geneknockout effect, which is in contrast to the relatively long-terminitiation and maintenance effects of D-RNAi. Also, the concentration ofmRNA-cDNA hybrids needed for enough biological effects is almost a halfmillion fold less than that of antisense DNAs. This suggests that theeffectiveness of D-RNAi is not the result of the cDNA part of amRNA-cDNA hybrid. It is likely that an RdRp-like enzyme generatesprecursors of small RNAs or aRNAs based on an mRNA template, therebymaintaining the relatively long-term D-RNAi effects.

There are three major effects of PTGS, i.e., initiation, spreading andmaintenance, all of which are also found in many inheritable RNAiphenomena. The initiation indicates that the onset of PTGS/RNAi takes arelatively long period of time (1˜3 days) to develop enough small RNAsor short aRNAs for specific gene knockout. With the antisensetransfection processes, it only takes several hours to reach the samegene silencing results but with much higher dosages and highercytotoxicity. Also, unlike the short-term effectiveness of traditionalantisense transfections, the PTGS/RNAi effects may spread from atransfected cell to neighboring cells and can be maintained for a verylong time (weeks to lifetime) in a mother cell as well as its daughtercells.

The results of the experiments here suggest that D-RNAi shares somefeatures of the PTGS/RNAi mechanisms. FIG. 4 shows a proposed model forlong-term PTGS/RNAi/D-RNAi mechanisms. Initiation and maintenanceperiods are varied, depending on different living systems andtransfected genes. Although the present invention is not limited tospecific mechanisms, the potential RdRp-dependent mechanism of D-RNAipossesses the initiation and maintenance, but not spreading features ofPTGS/RNAi effects. Because liposomal transfection methods offer only30˜40% transfection rate, a complete apoptosis induction in the LNCaPcell model used required at least two to three transfections (FIG. 5).FIG. 5 shows a linear plot of the interaction between incubation time(X) and cell growth number (Y), indicating no spreading effect of theD-RNAi. The black linear arrow shows the first addition of all testedprobes, while the dotted arrow indicates the second addition of anmRNA-cDNA probe for double transfection analysis of D-RNAi. Theproliferation rate of blank control (purple), aRNA-cDNA (blue) andds-RNA (green) transfected cells were not affected, whereas the growthof mRNA-cDNA (red and black) transfected cells remarkably inhibitedafter 36-hour incubation (n=4). Because one transfection is notsufficient to reach the entire cell population, a more completeinhibition of cell growth is achieved after double transfections(black), indicating no spreading effect of D-RNAi.

Identification of a Potential RdRp-Like Enzyme for D-RNAi in LNCaP Cells

RNA polymerase II has been found to possess RNA-directed RNA synthesisactivity (Filipovska et al., RNA 6: 41054 (2000); Modahl et al., Mol.Cell Biol. 20: 6030-6039 (2000)). Furthermore, the addition of low-doseα-amanitin (1.5 μg/ml), an RNA polymerase II-specific inhibitor derivedfrom a mushroom Amanita phalloides toxin, abrogated the apoptosisinduction of bcl-2 D-RNAi (FIG. 6).

FIG. 6 shows an analysis of a potential D-RNAi-related RdRp enzyme bydifferent α-amanitin sensitivity: (1) 1.5 μg/ml and (2) 0.5 μg/ml. FIG.6A shows the changes of cell proliferation rate and morphology afteraddition of α-amanitin. A significant reduction of D-RNAi-inducedapoptosis was detected in the 1.5 μg/ml α-amanitin addition (but not inthe 0.5 μg/ml α-amanitin addition) after mRNA-cDNA transfection (n=3),showing a dose-dependent release of cell growth inhibition. FIG. 6Bshows genomic laddering patterns demonstrating the blocking of theapoptotic induction effect of the bcl-2 mRNA-cDNA transfection by the1.5 μg/ml α-amanitin addition. FIG. 6C shows Northern blots indicatingthat the bcl-2 silencing effect of D-RNAi was prevented.

It seems that the RNA polymerase II or another α-amanitin-sensitiveRNA-directed polymerase is responsible for the RdRp activity of D-RNAiin human LNCaP cells. α-amanitin concentration up to 3.5 μg/ml can causepartial transcriptional inhibition without significant apoptosisinduction in the dihydrotestersterone-treated LNCaP cells. Although theα-amanitin concentration tested suppressed only about half transcriptionactivity, a remarkable inhibition of D-RNAi on bcl-2 has been detected,indicating that such potential RdRp enzyme is highlyα-amanitin-sensitive. According to the replication of HDV, which is alsoan RNA-directed RNA synthesis procedure, the binding of RNA polymeraseII to an RNA template requires an A-T rich domain but not necessarily aTATA-box as in the transcription of a DNA template.

Gene Silencing Using mRNA-cDNA Hybrids: In Vivo Model Targetingβ-Catenin in a Developing Chicken Embryo

The foregoing establishes that the novel mRNA-cDNA hybrids of thepresent invention can be used in a novel strategy to knock out targetedgene expression in vitro. As discussed below, the novel mRNA-cDNAstrategy of the invention is also effective in knocking out geneexpression in vivo.

As illustrated in the examples below, the methods and compositions ofthe invention are effective in knocking out targeted gene expression invivo in a developing chicken embryo. For molecules, β-catenin wastargeted because it has a critical role in development and oncogenesis,and for tissue, skin and liver were selected because the skin isaccessible and the liver is an important organ. β-catenin is known to beinvolved in the regulation of growth control. It has been suggested thatβ-catenin is involved in neovasculogenesis and that it may work withVE-cadherin, which is not essential for the initial endothelial adhesionbut is required in further vascular morphogenesis to properly formmature endothelial walls and blood vessels.

As discussed below, the experimental results establish that mRNA-cDNAhybrids are effective in inhibiting the expression of targeted genes,e.g., they potently inhibit β-catenin expression in the liver and skinof developing chick embryos. Thus, the results show that using anmRNA-cDNA duplex provides a powerful new strategy for gene silencing. AcDNA-aRNA duplex does not appear to work even though aRNA has beenpreviously shown to suppress gene expression. This may be due to the lowdosages used in the experiments here. However, this only underscores thefact that the mRNA-cDNA comprising compositions of the instant inventionare effective even at low dosages. The results also show that thisinvention is effective in knocking out the targeted gene expression overa long period of time (>10 days). Further, it was observed thatnon-targeted organs appear to be normal, which implies that thecompositions herein possess no overt toxicity. Thus, the inventionoffers the advantages of low dosage, stability, long term effectiveness,and lack of overt toxicity.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: M (molar); mM (millimolar); μm (micromolar); mol(moles); pmol (picomolar); gm (grams); mg (milligrams); L (liters); ml(milliliters); μl (microliters); °C. (degrees Centigrade); cDNA (copy orcomplimentary DNA); DNA (deoxyribonucleic acid); ssDNA (single strandedDNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotidetriphosphate); RNA (ribonucleic acid); PBS (phosphate buffered saline);NaCl (sodium chloride); HEPES(N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid); HBS (HEPESbuffered saline); SDS (sodium dodecylsulfate); Tris-HCl(tris-hydroxymethylaminomethane-hydrochloride); and ATCC (American TypeCulture Collection, Rockville, Md.).

All routine techniques and DNA manipulations, such as gelelectrophoresis, were performed according to standard procedures. (SeeSambrook et al., supra). All enzymes and buffer treatments were appliedfollowing the manufacture's recommendations (ROCHE BIOCHEMICALS,Indianapolis, Ind.). For Northern blots, mRNAs were fractionated on 1%formaldehyde-agarose gels and transferred onto nylon membranes(SCHLEICHER & SCHUELL, Keene, N H). Probes were labeled with thePrime-It II kit (STRATAGENE, La Jolla, Calif.) by random primerextension in the presence of [³²P]-dATP (>3000 Ci/mM, AmershamInternational, Arlington Heights, Ill.), and purified with MicroBio-Spin chromatography columns (BIO-RAD, Hercules, Calif.).Hybridization was carried out in the mixture of 50% freshly deionizedformamide (pH 7.0), 5×Denhardt's solution, 0.5% SDS, 4×SSPE and 250μg/ml denatured salmon sperm DNAs (18 h, 42° C.). Membranes weresequentially washed twice in 2×SSC, 0.1% SDS (15 min, 25° C.), and onceeach in 0.2×SSC, 0.1% SDS (15 min, 25° C.); and 0.2×SSC, 0.1% SDS (30min, 65° C.) before autoradiography.

Example 1 Cell Fixation and Permeabilization

LNCaP cells, a prostate cancer cell line, were grown in RPMI 1640 mediumsupplemented with 2% fetal calf serum. A sample containing cellscultured in a 60 mm dish (70% full of cells) was trypsinized, collectedand washed three times in 5 ml phosphate buffered saline (PBS, pH 7.2)at room temperature. After washing, the cells were suspended in 1 ml ofice-cold 10% formaldehyde solution in 0.15M NaCl. After one hourincubation on ice with occasional agitation, the cells were centrifugedat 13,000 rpm for 2 min, and washed three times in ice-cold PBS withvigorous pipetting. The collected cells were resuspended in 0.5% NonidetP40 (NP40, B.D.H.) and incubated for one hour with frequent agitation.The cells were washed three times in ice-cold PBS containing 0.1Mglycine, then resuspended in 1 ml of the same buffer with vigorouspipetting in order to be evenly separated into small aliquots and storedat —70° C. for up to a month.

Example 2 In-Cell Reverse Transcription and Poly-(N) Tailing of cDNAs

For reverse transcription of mRNAs in cells, twenty of the fixed cellswere thawed, resuspended in 20 μl of ddH₂O, heated to 65° C. for 3 minand then cooled on ice. A 50 μl RT reaction was prepared, comprising 5μl of 10× in-cell RT buffer (1.2M KCl, 0.5M Tris-HCl, 80 mM MgCl₂, 10 mMdithiothreitol, pH 8.1 at 42° C.), 5 μl of 5 mM dNTPs, 25 pmololigo(dT)n-T7 promoter, 80 U RNase inhibitor and above cold cells. Afterreverse transcriptase (40 U) was added, the RT reaction was mixed andincubated at 55° C. for three hours. The cells were then washed oncewith PBS and resuspended in a 50 μl tailing reaction, comprising 2 mMdGTP, 10 μl of 5× tailing buffer (250 mM KCl, 50 mM Tris-HCl, 7.5 mMMgCl₂, pH 8.3 at 20° C.). The tailing reaction was heated at 94° C. for3 min and then chilled in ice for mixing with terminal transferase (20U), following further incubation at 37° C. for 20 min. Final reactionwas stopped at 94° C. for 3 min. The reaction mixture was chilled in iceimmediately, which formed the poly(N)-tailed cDNAs.

Example 3

Single-Cell mRNA Amplification

To increase the intracellular copies of whole mRNAs, the T7 promoterregion of a poly(N)-tailed cDNA was served as a coding strand for theamplification by T7 RNA polymerase (Eberwine et al., Proc. Natl. Acad.Sci. USA 89: 3010-3014 (1992)). As few as one cell in 5 μl of abovetailing reaction can be used to accomplish full-length aRNAamplification. An in-cell transcription reaction was prepared on ice,containing 25 pmol poly(dC)-20mer primer, 1 mM dNTPs, Pwo DNA polymerase(5 U), 5 μl of 10× Transcription buffer (Boehringer Mannheim), 2 mM NTPsand T7 RNA polymerase (2000 U). The hybridization of 20mer primer to thepoly(N)-tailed cDNAs was incubated at 65° C. for 5 min to completesecond strand cDNA synthesis and then RNA polymerase was added to starttranscription. After four hour incubation at 37 ° C., the cDNAtranscripts were isolated from both cells and supernatant, to bedirectly used in the following reverse transcription. The reaction wasfinally stopped at 94° C. for 3 min and chilled in ice.

Example 4 In Vitro Reverse Transcription and PCR Amplification

A 50 μl RT reaction was prepared, comprising 5 μl of 10×RT buffer (300mM KCl, 0.5M Tris-HCl, 80 mM MgCl₂, 10 mM dithiothreitol, pH 8.3 at 20°C.), 5 μl of 5 mM dNTPs, 25 pmol oligo(dC)n-T7 promoter, 80 U Rnaseinhibitor, ddH₂O and 5 μl of the above aRNA containing supernatant.After reverse transcriptase (40 U) was added, the RT reaction wasvortexed and incubated at 55° C. for three hours. The resulting productsof RT can be directly used in following PCR reaction (50 μl), comprising5 μl of 10×PCR buffer (Boehringer Mannheim), 5 μl of 2 mM dNTPs, 25 pmolT7-20mer primer, 25 pmol poly(dT)-26mer primer, ddH₂O, 5 μl of above RTproduct and 3 U of Taq/Pwo long-extension DNA polymerase. The PCRreaction was subjected to thirty cycles of denaturation at 95° C. for 1min, annealing at 55° C. for 1 min and extension at 72° C. for 3 min.The quality of final amplified cDNA library (20 μl) was assessed on a 1%formaldehyde-agarose gel, ranging from 100 bp to above 12 kb.

Example 5 RNA-PCR

Pre-cycling procedures. Primers used in RNA-PCR were as follows: apoly(dT)24 primer (5′-TTTTTTTTTTTTTTTTTTTTTTTT-3′) (SEQ ID NO. 1) and anoligo(dC)₁₀N-promoter primer mixture comprising equal amounts ofoligo(dC)₁₀G-T7 primer (5′-dCCAGTGAAT TGTAATACGACTCACTATAGGGAAC₁₀G-3′)(SEQ ID NO. 2); oligo(dC)₁₀ A-T 7 primer(5′dCCAGTGAATTGTAATACGACTCACTATAGGGAAC₁₀ A-3′) (SEQ ID NO. 3); andoligo(dC)₁₀T-T7 primer (5′-dCCAGTGAATTGTAATACGACTCACTATAGGGAAC₁₀T-3′)(SEQ ID NO. 4). The poly(dT)₂₄ primer was used to reverse transcribemRNAs into first-strand cDNAs, while the oligo(dC)₁₀N-promoter primersfunctioned as a forward primer for second-strand cDNA extension from thepoly(dG) end of the first-strand cDNAs and therefore RNA promoterincorporation. All oligonucleotides were synthetic and purified by highperformance liquid chromatography (HPLC).

For in situ hybridization and cell preparations, fresh formaldehydeprefixed paraffin-embedded sections were dewaxed, dehydrated and refixedwith 4% PFA, and then permeabilized with protemase K (10 μg/ml; Roche)after rinsing with 1×PBS. In situ hybridization was achieved with adenatured hybridization mixture within a 200 μl coverslip chamber,containing 40% formamide, 5×SSC, 1×Denhardt's reagent, 50 μg/ml salmontestis DNA, 100 μg/ml tRNA, 120 pmol/ml poly(dT)₂₄ primer, 10 pmol/mlbiotin-labeled activin antisense probe (˜700 bases in size) and tissue.After 10 h incubation at 65° C., sections were washed once with 5×SSC at25° C. for 1 h and once with 0.5×SSC, 20% formamide at 60° C. for 30 minto remove unbound probes. A pre-heating step (68° C., 3 min) immersingthe sections in a mild denaturing solution (25 mM Tris-HCl, pH 7.0, 1 mMEDTA, 20% formamide, 5% DMSO and 2 mM ascorbic acid) was performed tominimize secondary structures (including crosslinks) and to reduce thebackground. After the temperature was lowered to 45° C.,2,5-diaziridinyl-1,4-benzoquinone (200 μM; Sigma Chemical Co., St Louis,Mo.) was added to each incubation for a further 30 min. Finally,0.1×SSC, 20% formamide was applied at 60° C. for 30 min to cleansections for chromogenic detection with straptavidin-alkalinephosphatase and Fast Red staining (Roche Biochemicals, Indianapolis,Ind.). Positive and negative results were observed and recorded under amicroscope. RNase-free enzymes and DEPC-treated materials were requiredthroughout the procedure.

Prostate cancer cells (20-150 cells) from in situ sections of patienttissues were isolated with a micromanipulator and directly used inRNA-PCR, while cultured LNCaP cells were preserved in 500 ld of ice-cold10% formaldehyde in suspension buffer (0.15 M NaCl pH 7.0, 1 mM EDTA)for the following fixation and permeabilization procedure. After 1 hincubation with occasional agitation, fixed LNCaP cells were collectedwith microcon-50 filters (Amicon, Beverly, Mass.) and washed with 350 μlof ice-cold PBS with vigorous pipetting. The collection and wash wererepeated at least once. The fixed cells were then permeabilized in 500μl of 0.5% NP-40 for 1 h with frequent agitation. After that, threecollections and washes were given to cells as before but using 350 μl ofice-cold PBS containing 0.1 M glycine instead. The cells were finallymixed with 0.1 μM poly(dT)₂₄ primer and resuspended in the same bufferwith vigorous pipetting to evenly distribute them into small aliquots(˜20 cells in 10 μl) for RNA-PCR. They could be stored at −80° C. for upto 2 weeks.

RNA-PCR. For amplification of intracellular mRNAs, more than 20 fixedcells were preheated at 94° C. for 5 min and applied to a reversetranscription (RT) reaction mixture (50 μl) on ice, comprising 10 μl of5×RT&T buffer [100 mM Tris-HCl, pH 8.5 at 25° C., 600 mM KCl, 300 mM(NH₄)₂SO₄, 25 mM MgCl₂, 5 M betaine, 35 mM dithiothreitol, 10 mMspennidine and 25% dimethylsulphoxide (DMSO)], 1 μM poly(dT)₂₄ primer,dNTPs (1 mM each dATP, dGTP, dCTP and dTTP) and RNase inhibitors (10 U).After 6 U Caxboxydothernius hydrogenoformans (C. therm.) polymerase(Roche) was added, the reaction was incubated at 52° C. for 3 min andshifted to 65° C. for another 30 min. The first-strand cDNAs so obtainedwere collected with a Microcon-50 microconcentrater filter, washed oncewith 1×PBS and suspended in a tailing reaction (50 μl ), comprising 10μl of 5×tailing buffer (250 mM KCl, 100 mM Tris-HCl, 4 mM CoCl₂, 10 mMMgCl₂, pH 8.3 at 20° C.) and 0.5 mM dGTP. After 75 U terminaltransferase (Roche) was added, the reaction was incubated at 37° C. for15 min, stopped by denaturation at 94° C. for 2 min and instantly mixedwith 1 μM oligo(dC)₁₀N-promoter primer mixture. After brieflycentrifuging, 3.5 U Taq DNA polymerase (Roche) and 1 mM of each of thedNTPs was added to form promoter-linked double-stranded cDNAs at 52° C.for 3 min, and then 72° C. for 7 min. The cells were broken by adding 1vol of 2% Nonidet P-40 (NP-40; Sigma Chemical Co.) for 10 min, and thenthe double-stranded cDNAs were washed and recollected with a microcon-50in autoclaved ddH₂O. This completed the pre-cycling steps for thefollowing cycling amplification.

A transcription reaction (50 μl) was prepared, containing 10 μl of5×RT&T buffer, rNTPs (1 mM each ATP, GTP, CTP and UTP), RNA inhibitors(10 U), T7 RNA polymerase (200 U; Roche) and the double-stranded cDNAs.After 2 h incubation at 37° C., the cDNA transcripts were isolated witha microcon-50 filter in 20 μl of DEPC-treated TE buffer (pH 7.0) andused directly for the next round of RNA-PCR without the tailingreaction, containing 10 μl of 5×RT&T buffer, 1 μM poly(dT)₂₄ primer, 1μM oligo(dC)₁₀N-promoter primers, dNTPs (1 mM each), rNTPs (1 mM each),C. therm. polymerase, Taq DNA polymerase and the transcription products(20 pg). T7 RNA polymerase was renewed in every transcription step dueto prior denaturation. The quality of mRNA products (20 μg) after threerounds of amplification was assessed on a 1% form aldehyde-agarose gel.

Northern blotting. mRNAs were fractionated on 1% formaldehyde-containingagarose gels and transferred to nylon membranes (Schleicher & Schuell,Keene, N. H.). Probes were labeled with the Pfime-It 11 kit (Stratagene,La Jolla, Calif.) by random primer extension in the presence of[³²P]dATP (>3000 Ci/mM; Amersham International, Arlington Heights,Ill.). Hybridization was carried out in the mixture of 50% freshlydeionized formamide (pH 7.0), 5×Denhardt's solution, 0.5% SDS, 4×SSPEand 250 μg/ml denatured salmon sperm DNA (18 h, 42° C.). Membranes werewashed twice in 2×SSC, 0.1% SDS (15 min, 25° C.), followed by once eachin 0.2×SSC, 0.1% SDS (30 min., 65° C.) before autoradiography.

Example 6 Thermostable Cycling Amplification Procedure

Few fixed and permeabilized cells were applied to a reaction mixture (20ml) on ice, comprising 2 ml of 10×RT&T buffer (400 mM Tris-HCl, pH 8.3at 25° C., 400 mM NaCl, 80 mM MgCl₂, 5M betaine, 100 mM DTT and 20 mMspermidine), 1 mM Shh-antisense primer, 1 mM Shh-sense promoter-primer,2 mM rNTPs, 2 mM dNTPs and RNase inhibitors (10 U). After C. therm./TaqDNA polymerase mixture (4 U) was added, the reaction was incubated at52° C. for 3 min, at 65° C. for 30 min, at 94° C. for 3 min, at 52° C.for 3 min, and then at 68° C. for 3 min. A transcription reaction wasprepared by adding T7 RNA polymerase (200 U) and C. therm. polymerase (6U) mixture into above reaction. After one hour incubation at 37° C., theresulting mRNA transcripts were continuously reverse-transcribed intomRNA-cDNA hybrids at 52° C. for 3 min, and then at 65° C. for 30 min.The quality of amplified mRNA-cDNA products can be assessed on a 1%formaldehyde-agarose gel (Lin et al., Nucleic Acid Res. (1999)).

Example 7 Liposomal Transfection Procedure

An mRNA-cDNA hybrid Shh probe (10 mg) was dissolved in 75 ml of Hepesbuffer (pH 7.4). The resulting solution was mixed with 50 ml of DOTAP®liposome (1 mg/ml, Roche Biochemicals) on ice for 30 min., thensubsequently applied to 60 mm diameter culture dishes containing four orfive chicken skin explants. The skin explants were grown in HBSS medium.After a 36 hour incubation, the disturbance of feather growth wasobserved only in the mRNA-cDNA hybrid set while the blank-liposomal andcDNA-aRNA hybrid control have no effects (FIG. 8A). The Northern blotresults of blank control and mRNA-cDNA hybrid set showed that a 73% genesilencing effect occurred by treating the mRNA-cDNA hybrid Shh probes(FIG. 8B).

Example 8 Gene Silencing Using a Chicken Embryo Model

This Example shows the effectiveness of an mRNA-cDNA strategy toknockout gene expression in vivo, using a developing chicken embryo as amodel. In this example, β-catenin expression was targeted in the skinand liver of developing chick embryos. The mRNA-cDNA duplexes used forknocking β-catenin expression in vivo can be generated using theimproved RNA-PCR technology discussed above.

For β-catenin, a double-stranded DNA template fragment, a pair ofprimers was designed based on the cDNA sequence. The central region forantisense targeting of β-catenin (aa 306 -644) required four primers(i.e., primers A-D). The upstream (A) primer comprises the sequence5′-ATGGCAATCAAGAAAGTAAGC-3′ (SEQ ID. NO. 5). The downstream (B) primercomprises the sequence 5′-GTACAACAACTGCACAAATAG-3′ (SEQ ID. NO. 6).Another set of primers was required for the generation of the desiredduplexes. The (C) primer was generated by adding the T7 promoter (RP)before the 5′ end of the (A) primer. The (D) primer was generated byadding the T7 promoter before the 5′ end of the (B) primer.

For mRNA-cDNA templates, B and C primers were used as primers in apolymerase chain reaction to generate promoter-linked double strandedcDNA. The promoter-linked double stranded cDNA was transcribed with T7RNA polymerase for 2 h, and AMV reverse transcriptase for 1 hourSubsequently, the DRNAi hybrids were collected by filtration over aMicrocon 50 (Amicon, Bedford, Mass.) column and eluted with 20 μl ofelution buffer (20 mM HEPES). The final concentration of DRNAi isapproximately 25 nM.

For the cDNA-aRNA template, A and D primers were used in a similarprocedure as described above in the opposite orientation. The size ofthe hybrids was then determined on a 1% agarose gel. The hybrids werekept at −20° C. until use.

Fertilized eggs were obtained from SPAFAS farm (Preston, Conn.) andincubated in humidified incubator (Humidaire, New Madison, Ohio). Atdesignated dates, eggs were put under a dissection microscope and theegg shells were sterilized. The shells were carefully cracked open and awindow was made to get access to the embryos.

Using embryonic day three chicken embryos, either mRNA-cDNA or cDNA-aRNA(25 nM) was injected into the ventral body cavity, close to where theliver primordia would form. The mRNA-cDNA hybrid was mixed with DOTAP®liposome (Roche, Indianapolis, Ind.) at a ratio of 3:2. A 10% (v/v) fastgreen solution was added before the injection to increase visibility(FIG. 9B). The mixtures were injected into the ventral side near theliver primordia and below the heart using heat pulled capillary needles.After injection, the eggs were sealed with scotch tape and put back intoa humidified incubator (Lyon Electric Company, Chula Vista, Calif.) at39-40° C. until the harvesting time.

At designated days after injection, the embryos were removed, examinedand photographed under a dissection microscope. While there aremalformations, the embryos survived and there was no overt toxicity oroverall perturbation of embryo development. The liver was closest to theinjection site and is most dramatically affected in its phenotypes.Other regions, particularly the skin, are also affected by the diffusednucleotides.

Selected organs were removed and total RNAs were collected with anRNeasy kit (QIAGEN, Valencia, Calif.) for Northern analysis. RNAs werefractionated in an RNase free polyacrylamide gel (1%) and thentransferred to Nylon membranes for 16-18 h. The tested gene washybridized with a radiolabeled probe, and an autoradiograph was exposed.Northern blot hybridizations using RNA from dissected livers showed thatβ-catenin in the control livers remained expressed (lane 4-6, FIG. 9C),whereas the level of β-catenin mRNA was decreased dramatically (lane1-3, FIG. 9D) after treatment with DRNAi directed against β-catenin. Inthis figure, C is hybridized to a β-catenin probe, while D is hybridizedto a GAPDH probe, to show that equivalent concentrations were loaded.Controls used include liposome alone and similar concentrations ofcDNA-aRNA.

Livers after ten days of injection with mRNA-cDNA duplex showed anenlarged and engorged first lobe, but the size of the second and thirdlobes of the livers were dramatically decreased (FIG. 10A-A′).Histological sections of normal liver showed hepatic cords andsinusoidal space with few blood cells. In the β-catenin treated embryos,the general architecture of the hepatic cells in lobes 2 and 3 remainedunchanged. However, in lobe 1 there are islands of abnormal regions. Theendothelium development appears to be defective and blood is outside ofthe blood vessels. Abnormal types of hematopoietic cells are observedbetween the space of hepatocytes, particularly dominated by a populationof small cells with round nuclei and scanty cytoplasm. In severelyaffected areas, hepatocytes were disrupted (FIG. 10B, B′).

Since skin is exposed in the amniotic cavity and is most accessible tothe nucleotides that leaked out, patches of skin that showed phenotypeswere also observed. At embryonic day 13, skin should have formedelongated feather buds, with a primordial blood vessel running into itsmesenchymal core. In the mRNA-cDNA β-catenin affected region, featherbuds become engorged with blood, starting from the distal end of thefeather tip (FIG. 10C, C′). The adjacent skin was normal (not shown),and works as a good control. Histological sections showed that thenormal feather buds have continued their morphogenetic process with theepidermis invaginated to form the feather follicle walls, surrounding amesenchymal core. In affected areas, the distal feather bud mesenchymewas full of engorged blood vessels and blood cells. Distal epidermisalso detached from the feather mesenchyme, and proximal epidermis failedto invaginate to form follicles (FIG. 10D, D′).

Example 9 Generation of bcl-2 RNA-DNA Hybrids

Four synthetic oligonucleotides were used in the generation of bcl-2RNA-DNA hybrids as follows: T7-bcl2 primer(5′-dAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGGATGACTGAGTACCTGAACCGGC-3′)(SEQ ID. NO. 7) and anti-bcl2 primer (5′-dCTTCTTCAGGCCAGGGAGGCATQG-3′)(SEQ ID. NO. 8) for mRNA-cDNA hybrid (D-RNAi) probe preparation;T7-anti-bcl2 primer(5′-dAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCCTTCTTCAGGCCAGGGAGGCATGG-3′)(SEQ ID NO. 9) and bcl2 primer (5′-dGGATGACTGAGTACCTGAACCGGC-3′) (SEQ IDNO. 10) for antisense RNA (aRNA)-cDNA hybrid (reverse D-RNAi) probepreparation. The design of the sequence-specific primers is based on thesame principle used by PCR (50˜60% G-C rich), while that of thepromoter-linked primers however requires a higher G-C content (60˜65%)working at the same annealing temperature as above sequence-specificprimers due to their unmatched promoter regions. For example, newannealing temperature for the sequence-matched region of apromoter-linked primer is equal to [2° C.×(dA+dT)+3° C.×(dC+dG)]×5/6,not including the promoter region. All primers were purified bypolyacrylamide gel electrophoresis (PAGE) before use in RNA-PCRreaction.

Example 10 Treatment of LNCaPCells to Induce bcl-2 Expression

LNCaP cells were obtained from the American Type Culture Collection(ATCC, Rockville, Md., and grown in RPMI 1640 medium supplemented with10% fetal bovine serum with 100 μg/ml gentamycin at 37° C. under 10%CO₂. These cultured cells were treated with one dose of 100 nM5α-anrostan-17β-ol-3-one to induce bcl-2 expression. For liposomaltransfection of anti-bcl-2 probes, the probes (5 nM) in DOTAP liposome(Roche Biochemicals) were applied to a 60 mm culture dish whichcontained LNCaP cells at 15% confluency. After a 18-hour incubation, thecells took up about 60% of the probe-containing liposome. Uptakeimproved to 100% after 36 hours of incubation. The addition ofα-amanitin was completed at the same time as the liposomal transfection.The apoptotic effect of phorbol-12-myristate -13-acetate (10 mM) wasinitiated at 12 hours after liposomal transfection. The mRNAs from thetransfected LNCaP cells were isolated by poly-(dT) dextran columns(Qiagen, Santa Clarita, Calif.), fractionated on a 1%formaldehyde-agarose gel after a 36-hour incubation period, andtransferred onto nylon membranes. After 48-hour transfection, genomicDNAs were isolated by an apoptotic DNA ladder kit (Roche Biochemicals)and assessed on a 2% agarose gel. Cell growth and morphology wereexamined by microscopy and cell counting, following known techniques.(See e.g., Lin et al., Biochem. Biophys. Res. Commun. 257: 187-192(1999)).

Example 11 Probe Preparations from Androgen-Treated LNCaP Cells

For the generation of RNA-DNA hybrid probes, an RNA-polymerase cyclingreaction (RNA-PCR) procedure was modified to generate either mRNA-cDNAor cDNA-aRNA hybrids. Total RNAs (0.2 μg) from androgen-treated LNCaPcells were applied to a reaction (50 μl in total) on ice, comprising 5μl of 10×RT&T buffer (400 mM Tris-HCl, pH 8.3 at 25° C., 400 mM NaCl, 80mM MgCl₂, 2 M betaine, 100 mM DTT and 20 mM spermidine), 1 μMsequence-specific primer for reverse transcription, 1 μM promoter-linkedprimer for cDNA-doublestranding, 2 mM rNTPs, 2 mM dNTPs and RNaseinhibitors (10 U). After C. therm./Taq DNA polymerase mixture (4 U each)was added, the reaction was incubated at 52° C. for 3 min, 65° C. for 30min, 94° C. for 3 min, 52° C. for 3 min and then 68° C. for 3 min. Thisformed a promoter-linked double-stranded cDNA for next step oftranscriptional amplification up to 2000 fold/cycle. An in-vitrotranscription reaction was performed by adding T7 RNA polymerase (160 U)and C. therm. polymerase (6 U) into above reaction. After one hourincubation at 37° C., the resulting mRNA transcripts were continuouslyreverse-transcribed into mRNA-cDNA hybrids at 52° C. for 3 min and then65° C. for 30 min. The generation of cDNA-aRNA hybrids was the sameprocedure as aforementioned except using 1 μM sequence-specific primerfor cDNA-doublestranding and 1 μM promoter-linked primer for reversetranscription. The RNA-PCR procedure can be reiterated to produce enoughRNA-DNA hybrids for gene silencing analysis. For the preparation ofdouble-stranded RNA probes, complementary RNA products were transcribedfrom both orientations of above promoter-linked double-stranded cDNAsand mixed together without reiterating reverse transcription activity.The quality of amplified probes were assessed on a 1%formaldehyde-agarose gel.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art, andare to be included within the spirit and purview of the invention as setforth in the appended claims. All publications and patents cited hereinare incorporated herein by reference in their entirety for all purposes.

1. A method for inhibiting the expression of a target gene through apost-transcriptional gene silencing mechanism in a cell that expressesthe targeted gene, comprising the steps of: a) providing a compositioncomprising an mRNA-cDNA hybrid duplex prior to contacting said cell,wherein the mRNA-cDNA hybrid duplex is capable of inhibiting theexpression of said targeted gene in said cell, wherein said expressionis inhibited through said post-transcriptional gene silencing mechanism,wherein said targeted gene is a β-cantenin gene, and wherein said cellis in the liver or skin of a chicken embryo; and b) contacting said cellwith said composition under conditions such that the expression of saidgene in said cell is inhibited, wherein the mRNA is a ribonucleic acidsequence in the sense orientation of said targeted gene and the cDNA isa deoxyribonucleic acid sequence in the anti-sense orientation of saidtargeted gene, and wherein the mRNA-cDNA hybrid duplex forms betweensaid mRNA and said cDNA in a complementary region containing more than500 base pairs.
 2. The method of claim 1, wherein said mRNA-cDNA hybridduplex inhibits the expression of said targeted gene, wherein saidtargeted gene comprises a β-catenin sequence encoding its amino aciddomain from position 306 to
 644. 3. The method of claim 1, wherein thecomposition consists of an mRNA-cDNA hybrid duplex capable of inhibitingthe expression of said targeted gene, wherein the mRNA is a ribonucleicacid sequence in the sense orientation of said targeted gene and thecDNA is a deoxyribonucleic acid sequence in the anti-sense orientationof said targeted gene, wherein the mRNA-cDNA hybrid duplex forms betweensaid mRNA and said cDNA in a complementary region containing more than500 base pairs.
 4. The method of claim 3, wherein said mRNA-cDNA hybridduplex inhibits the expression of said targeted gene, wherein saidtargeted gene comprises a β-catenin sequence encoding its amino aciddomain from position 306 to
 644. 5. A method for inhibiting theexpression of a target gene through a post-transcriptional genesilencing mechanism in a cell that expresses the targeted gene,comprising the steps of: a) providing a composition comprising anmRNA-cDNA hybrid duplex prior to contacting said cell, wherein themRNA-cDNA hybrid duplex is capable of inhibiting the expression of saidtargeted gene in said cell, wherein said expression is inhibited throughsaid post-transcriptional gene silencing mechanism, wherein saidtargeted gene is a β-cantenin gene, and wherein said cell is in theliver or skin of a chicken embryo; and b) contacting said cell with saidcomposition under conditions such that the expression of said gene insaid cell is inhibited, wherein the mRNA is a ribonucleic acid sequencein the sense orientation of said targeted gene and the cDNA is adeoxyribonucleic acid sequence in the anti-sense orientation of saidtargeted gene, and wherein the mRNA is a full-length transcript of saidtargeted gene larger than 500 base pairs.
 6. The method of claim 5,wherein the mRNA is an unspliced mRNA transcript of the targeted gene.7. The method of claim 5, wherein the mRNA is a spliced mRNA transcriptof the targeted gene.